1. Field of the Invention
The present invention relates to the use of a proteineous component isolated from plant chromatin. More precisely, the invention relates to the use of a proteineous component isolated from plant chromatin, after dissociation of the same, as an antimicrobial agent, as well as a method of producing the same.
2. Related Background Art
Most eukaryotic organisms produce a wide variety of protective mechanisms directed towards infectious agents. Several mechanisms are based on those fundamental differences, which exist in membrane composition and organization between microbes and cells of complex multicellular organisms, i.e. they are directed towards outer membranes of sensitive microbes. These membranes are composed of lipids having negatively charged head groups facing outwards, and the microbes apparently find it difficult to counteract the effects by altering their membrane composition and organization. Thus, the substances responsible for the antibiotic action are presumable candidates as substitutes for antibiotics.
One example is the phospholipid transfer proteins, which are able to transfer phospholipids between membranes. Antimicrobial phospholipid transfer proteins have been reported from a range of plant species including cereals, and these proteins vary in their activity against different pathogens. For example, in U.S. Pat. No. 5,698,200 it is shown that a plant part can be protected from a plant pathogenic bacterium by means of an aqueous extract obtained from malted cereal grain.
However, the most studied class of protective agents is the antimicrobial peptides. They are found in all species of life, ranging from plants and insects to animals, including molluscs, crustaceans, amphibians, birds, fish, mammals, and humans.
These peptides interact directly with bacteria and kill them. They are termed antimicrobial because they have unusually broad spectra of activity including the ability to kill or neutralize Gram-negative and Gram-positive bacteria fungi (including yeast), parasites (including planaria and nematodes), cancer cells, and even enveloped viruses like HIV and herpes simplex virus. In general, these agents range in length from as few as 12 amino acids to molecules with over 70 residues. More than 500 such peptides have been discovered.
The mode of antimicrobial action of the almost always cationic antimicrobial peptides has been studied in detail among such peptides as melittin, magainin, gramicidin, cecropin, and defensins. The antimicrobial molecules also generally damage the membranes of the organisms that they attack. The cationic antimicrobial peptides have been found to possess bactericidal activity in vitro as well as in vivo. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, is other peptides as well as lysozyme, and are able to kill bacteria in animal models.
As a consequence, antimicrobial peptides of animal origin are now developed as new antibiotic drug. Examples are the synthetic version of magainin (pexiganan) and the analogue of a protegrin, an antimicrobial peptide initially isolated from pig neutrophils.
However, natural sources have not proved to be economically profitable for the production of new alternative antibiotics. The only exception is the antimicrobial peptide nisin, which can be effectively produced in a Lactococcus lactis strain with high resistance to the substance.
An increasing number of larger proteins or fragments thereof have also been found to exhibit antimicrobial activities. For example, a murine macrophage protein, ubiquicidin, appears to be the same as the ribosomal protein S30. Also, two of the antimicrobial peptides in the stomach of bullfrog (Rana catesbeina) are derived from the N-terminus of pepsinogen. Likewise, an antimicrobial peptide, named buforin I, has been isolated from stomach tissue of an Asian toad (BBRC 218:408, 1996). The amino acid sequence of the 39 amino acid long peptide was found to be identical with 37 of the 39 amino-terminal residues of the Xenopus histone H2A.
In addition, the whole protein molecule can exhibit an antimicrobial potential. Antimicrobial activity has been detected in acid extracts of liver, intestine, and stomach of atlantic salmons (BBRC 284:549, 2001): The corresponding antimicrobial protein can be isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel chromatography (gel filtration), reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 27.7 kD and was identified as the histone H1 protein. In WO 200110901, the mammalian histone H1 protein from bovine thymus is used in antimicrobial compositions for treating microbial infections in different eucaryotic organisms. Thus, proteins having other well-established functions appear to exhibit a second property by being antimicrobial.
However, the use of bovine proteins, especially proteins from bovine thymus, should be avoided since such a material can be contaminated with deleterious virus, especially hepatitic viruses, or other pathogenic agents, for example priones. Bovine material—whether contaminated or not—must be subjected to extremely strict tests when intended to be used in connection with humans.
Furthermore, the isolation of new alternative antibiotics involves the collection of specified animal organs or tissue, followed by complex purification procedures in order to obtain a product that can be used in connection with human beings or domestic animals.